Project Summary Humans have limited regenerative potential after extensive musculoskeletal injuries, particularly following limb loss. As such, the ability to biologically restore the missing limb will significantly improve the quality of life for millions of amputees. To understand the fundamental mechanisms guiding regeneration of complex musculoskeletal tissues or organs, mouse models of digit amputation have been used to study musculoskeletal regrowth, which occurs in a spatiotemporally controlled manner upon distal digit tip removal. While much progress has been made to elucidate the cell types and growth factors responsible for this natural regenerative response, the exact cellular and molecular mechanisms underlying digit regeneration have yet to be revealed. Recent studies suggest that cells derived from the periosteum participate in bone regrowth in adults, and thus represent a possible source of osteoblast lineage cells. However, it remains unknown to what extent this specific cell population plays a direct role in digit regeneration. We hypothesize that spatiotemporal activation of distinct molecular events after distal digit amputation mediates the activation, proliferation, and/or differentiation of periosteal cells (PCs). To test this hypothesis, we will investigate the spatiotemporal response of PCs after digit amputation using Osx-CreERT2;Ai9 inducible reporter transgenic mice in Aim 1, which will permit lineage tracing of this cell population. Furthermore, we will test the requirement of proliferating PCs during digit regeneration by inducible conditional ablation of replicating PCs using 3.6Col1a1-tk transgenic mice. Finally, we will determine the signaling cues that activate and sustain digit regeneration in Aim 2 using RNA in situ hybridization and tissue-specific RNA-sequencing, with a specific focus on the spatiotemporal distribution of canonical Wnt signaling. By identifying the cells and molecular pathways vital to regeneration, this proposal will open new avenues to therapies that will help restore the biological composition and structure of the lost tissues.